The efficacy of synaptic transmission varies greatly among synaptic contacts. We have explored the origins of differences between phasic and tonic crustacean neuromuscular junctions. Synaptic boutons of a phasic motor neuron release three orders of magnitude more quanta to a single action potential and show strong depression to a train, whereas tonic synapses are nearly unresponsive to single action potentials and display an immense facilitation.

We tested the hypothesis that the probability of vesicular exocytosis at synapses is positively correlated with the pools of readily releasable synaptic vesicles, as shown for mammalian neurons grown in tissue culture. We compared synapses of two identified glutamatergic neurons: phasic (high-output, depressing) and tonic (low-output, facilitating) crustacean motor neurons, which differ 100- to 1000-fold in quantal content. Estimates of vesicles available for exocytosis were made from depletion during forced release and from electron microscopic observation of vesicles docked at synaptic membranes near active zones. Both measurements showed a significantly larger pool of readily releasable vesicles in facilitating synapses, despite their much lower quantal output during stimulation. Thus, the probability for release of docked vesicles is very much lower at facilitating synapses, and the presence of more docked vesicles does not predict higher synaptic release probability in these paired excitatory neurons.

Synaptic functional differentiation of crayfish phasic and tonic motor neurons is large. For one impulse, quantal release of neurotransmitter is typically 100-1000 times higher for phasic synapses. We tested the hypothesis that differences in synaptic strength are determined by differences in synaptic calcium entry. Calcium signals were measured with the injected calcium indicator dyes Calcium Green-1 and fura-2. Estimated Ca(2+) entry increased almost linearly with frequency for both axons and was two to three times larger in phasic terminals. Tonic terminal Ca(2+) at 10 Hz exceeded phasic terminal Ca(2+) at 1 Hz, yet transmitter release was much higher for phasic terminals at these frequencies. Freeze-fracture images of synapses revealed on average similar numbers of prominent presynaptic active zone particles (putative ion channels) for both neurons and a two- to fourfold phasic/tonic ratio of active zones per terminal volume. This can account for the larger calcium signals seen in phasic terminals. Thus, differences in synaptic strength are less closely linked to differences in synaptic channel properties and calcium entry than to differences in calcium sensitivity of transmitter release.

Crustacean motor neurons subserving locomotion are specialized for the type of activity in which they normally participate. Neurons responsible for maintained activity ('tonic' neurons) support moderate to high frequencies of nerve impulses intermittently or continuously during locomotion, while those recruited for short-lasting rapid responses ('phasic' neurons) generally fire a few impulses in a rapid burst during rapid locomotion and are otherwise silent. The synaptic responses of the two types, recorded at their respective neuromuscular junctions, differ enormously: phasic neurons exhibit much higher quantal release per synapse and per muscle fibre, along with more rapid synaptic depression and less short-term facilitation. We have analyzed the factors that are responsible for the large difference in initial release of neurotransmitter. Several possibilities, including synapse and active zone size differences, entry of calcium at active zones, and immediately releasable vesicle pools, could not account for the large phasic-tonic difference in initial transmitter output. The most likely feature that differentiates synaptic release is the sensitivity of the exocytotic machinery to intracellular calcium. Molecular features of the phasic and tonic presynaptic nerve terminals are currently under investigation.